Optical control of acetylcholinesterase with a tacrine switch

Article abstract of DOI:10.1002/anie.201403666

Photochromic ligands have been used to control a variety of biological functions, especially in neural systems. Recently, much effort has been invested in the photocontrol of ion channels and G-protein coupled receptors found in the synapse. Herein, we describe the expansion of our photopharmacological approach toward the remote control of an enzyme. Building on hallmark studies dating from the late 1960s, we evaluated photochromic inhibitors of one of the most important enzymes in synaptic transmission, acetylcholinesterase (AChE). Using structure-based design, we synthesized several azobenzene analogues of the well-known AChE inhibitor tacrine (THA) and determined their effects on enzymatic activity. One of our compounds, AzoTHA, is a reversible photochromic blocker of AChE in vitro and ex vivo with high affinity and fast kinetics. As such, AzoTHA can be used to control synaptic transmission on the neuromuscular endplate based on the light-dependent clearance of a neurotransmitter.

Full text of DOI:10.1002/anie.201403666

Angewandte
Chemie
DOI: 10.1002/anie.201403666
Photopharmacology
Optical Control of Acetylcholinesterase with a Tacrine Switch**
Johannes Broichhagen, Innokentij Jurastow, Katharina Iwan, Wolfgang Kummer, and
Dirk Trauner*
Abstract: Photochromic ligands have been used to control
a variety of biological functions, especially in neural systems.
Recently, much effort has been invested in the photocontrol of
ion channels and G-protein coupled receptors found in the
synapse. Herein, we describe the expansion of our photo-
pharmacological approach toward the remote control of an
enzyme. Building on hallmark studies dating from the late
1960s, we evaluated photochromic inhibitors of one of the most
important enzymes in synaptic transmission, acetylcholinester-
ase (AChE). Using structure-based design, we synthesized
several azobenzene analogues of the well-known AChE
inhibitor tacrine (THA) and determined their effects on
enzymatic activity. One of our compounds, AzoTHA, is
a reversible photochromic blocker of AChE in vitro and
ex vivo with high affinity and fast kinetics. As such, AzoTHA
can be used to control synaptic transmission on the neuro-
muscular endplate based on the light-dependent clearance of
a neurotransmitter.
ase (AChE), which hydrolyzes ACh to acetate and choline
with diffusion-controlled kinetics (Figure 1a).[2] A large
number of AChE inhibitors are known, which range from
drugs, such as tacrine (THA), to research tools, such as
decamethonium, and nerve gases, such as sarin (Figure 1b).
Many of these have been co-crystallized with AChE, provid-
ing detailed insights into the mode of action of this enzyme
and its inhibitors.[3] Some of these are clinically relevant,
because they raise ACh levels, which has beneficial effects to
patients suffering, for instance, from Alzheimerꢀs disease,
myasthenia gravis, and glaucoma.[4]
In recent years, the control of neural systems with light has
become a major scientific frontier. Light is noninvasive and
can be applied with very high temporal and spatial control. It
can interact with genetically encoded photoreceptors (“opto-
genetics”)[5] or with photoswitchable drugs that target native
receptors and change their efficacy upon photoisomerization
(Figure 1c). We call the latter approach “photopharmacol-
ogy”.[6] It has been applied to the optical control of ion
channels[7] and GPCRs[8] and has been used to restore visual
response in blind mice[9] and control nociception mediated by
DRG neurons.[10]
Enzymes such as AChE, which have fast kinetics and
a large influence on the activity of neural networks, are also
a prime target for photopharmacology. Indeed, AChE has
been investigated in this respect early on. Pioneering studies
from the late 1960s and early 1970s by Erlanger and
colleagues show that photoregulation of AChE could be
achieved with azobenzene-based quaternary ammonium ions
that mimic ACh, such as p-phenylazophenyl-trimethylammo-
nium and N-p-phenylazophenylcarbamylcholine (Fig-
ure 1d).[11] Photocontrol of an AChE isolated from the
scorpion Heterometus fulvipes was achieved by exposure to
sunlight.[11b] Activity was measured by a reaction between
nonhydrolyzed ACh and hydroxylamine, followed by photo-
metric quantification of an iron complex,[12] thus providing
a proof-of-concept for a photoswitchable enzyme inhibitor.
Since Erlangerꢀs pioneering studies, the ability to hetero-
logously express enzymes, reliably assay them, study them
with X-ray crystallography, and rationally design ligands has
much improved. In addition, the precise delivery of light of
a given wavelength and intensity has become more practical,
recently driven by the rapid development of optogenetics.
Given our longstanding interest in the optical control of
neural systems, we therefore decided to reinvestigate photo-
chromic blockers of mammalian AChE, that is, human AChE,
and to explore new chemotypes that could prove to be useful
in neuroscience.
S
ynaptic communication is largely based on small diffusible
molecules that translate electrical signals into chemical
ones.[1] Once released from synaptic vesicles, these neuro-
transmitters cross the synaptic cleft to stimulate receptors,
that is, ligand-gated ion channels and G-protein coupled
receptors, on the postsynaptic side. A third essential compo-
nent of synaptic transmission consists of transporters or
enzymes that remove or inactivate the neurotransmitter,
respectively, to prevent tonic stimulation and allow for the
meaningful integration of signals.
Among a diverse set of neurotransmitters that mediate
chemical communication in humans, acetylcholine (ACh) is
especially important. When released, it stimulates nicotinic
and muscarinic acetylcholine receptors to modulate cellular
excitability. Fast inactivation is achieved by acetylcholinester-
[*] Dipl.-Chem. J. Broichhagen, K. Iwan, Prof. Dr. D. Trauner
Department Chemie, Ludwig-Maximilians-Universitꢀt Mꢁnchen
und Center of Integrated Protein Science Munich
Butenandtstrasse 5–13, 81377 Mꢁnchen (Germany)
E-mail: dirk.trauner@lmu.de
I. Jurastow, Prof. Dr. W. Kummer
Institut fꢁr Anatomie und Zellbiologie
Justus-Liebig-Universitꢀt Gießen
German Center for Lung Research
Aulweg 12335392 Gießen (Germany)
[**] J.B. is grateful to the Studienstiftung des deutschen Volkes for
a Ph.D. Fellowship. We thank M. Schçnberger and D. H. Wood-
mansee for helpful discussions and excellent advice.
Supporting information for this article (experimental details
including all synthesis, chemical characterizations, and assay
conditions) is available on the WWW under http://dx.doi.org/10.
1002/anie.201403666.
In order to quickly assay human AChE activity in the
presence of a photoswitchable inhibitor, we modified a com-
mercially available kit, which is used to measure the AChE
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resemblance with decamethonium, we won-
dered whether it could also act as a photo-
chromic inhibitor of the enzyme as well. We
found that BisQ is indeed a blocker of
human AChE in the dark (Ki = 3.13 mm,
Table 1), but does not significantly change
its activity upon photoisomerization. This
result and an inspection of the X-ray
structure of decamethonium bound to
AChE (pdb: 1ACL)[13] imply that BisQ
does not simultaneously occupy both cat-
ionic binding sites when blocking the
enzyme. Thus we hypothesized that both
isomeric forms of BisQ exhibit comparable
blocking characteristics.
A systematic exploration of AChE X-
ray structures showed that the inhibitors
tacrine (THA; pdb: 1ACJ)[13] and 9-benzyl
THA (pdb: 1DX4)[16] occupy the catalytic
site of the enzyme, while being partially
solvent-exposed. The knowledge that sub-
stitution in the 9-position of THA is tol-
erated enabled the design of several azo-
benzene derivatives that we call AzoTHAs
(Scheme 1a). Our small series includes
Figure 1. AChE function and inhibition. a) AChE (pdb: 1ACJ)[13] catalyzes the hydrolysis of
acetylcholine (ACh) to give acetate and choline. b) Noncovalent and covalent inhibitors of
AChE activity. c) Photopharmacological control of an enzyme with a photochromic inhibitor.
d) Photochromic molecules used to control the cholinergic system.
a
minimally substituted azobenzene
(AzoTHA), an ester derivative thereof
(AzoTHA-2), and a triazole (AzoTHA-3),
which was prepared by “click chemistry”
to pave the way for rapid diversification.
The synthesis of AzoTHA started with the
condensation of anthranilic acid (1) and
cyclohexanone (2) in refluxing phosphoryl
chloride to give 9-chloro-THA (3).[17] Sub-
stitution of the chloride with 4-(2-amino-
ethyl)aniline (4) enabled by catalytic
amounts of sodium iodide in refluxing
phenol gave phenethylamine derivative 5.
Finally, the azobenzene moiety was instal-
led through Mills reaction using nitro-
sobenzene (6; Scheme 1b). As a “regular”
azobenzene, AzoTHA could be switched
to its cis form with UV light (l = 350 nm)
and back to its trans form using blue light
(l = 440 nm; Scheme 1b,c). The small
change in the p–p* band upon irradiation
Figure 2. Colorimetric assay for measuring the AChE activity in the presence of a photo-
switchable blocker. a) Hydrolysis of acetylthiocholine gives thiocholine, which reacts with
Ellman’s reagent to give a colored thiolate (lmax =410 nm). b) Schematic representation of
the experimental configuration.
content in a variety of biological samples.[14] It utilizes the
intrinsic ability of AChE to cleave acetylthiocholine, which
subsequently reacts with Ellmanꢀs reagent to liberate a col-
ored thiolate (Figure 2a). Using a UV/Vis spectrometer, we
were able to photometrically follow the appearance of the
thiolate at l = 410 nm, while alternately irradiating with an
orthogonal light beam of different wavelengths, to reversibly
trigger photoswitching (Figure 2b).
can be partly attributed to the underlying absorption of the
quinoline chromophore of THA (black line, Scheme 1c).
Details of the switching kinetics of AzoTHA and synthesis of
AzoTHA-2 and AzoTHA-3 can be found in the Supporting
Information.
AzoTHA in its dark-adapted form inhibits AChE with
a Ki of approximately 100 nm (Table 1), while AzoTHA-2 and
Our initial efforts were directed at the doubly charged
ACh mimic BisQ (Figure 1d), which was developed by
Erlanger and Lester as a photoswitchable agonist for the
nicotinic acetylcholine receptor,[15] but had not been de-
scribed as photoswitchable inhibitor of AChE. Given its
Table 1: Inhibitory constants (Ki) of AChE blockers (N=3, meanÆ
standard deviation).
THA
AzoTHA
decamethonium BisQ
3129.3Æ1181.4
Ki [nm] 6.40Æ3.20 95.35Æ12.39 457.8Æ52.0
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neuromuscular endplate, in an experimen-
tal setting similar to the classical Loewi
experiment.[19] When the electrical stimu-
lation is shut off, ACh release from nerve
fibers ceases and the relaxation kinetics of
the trachea reflect AChE activity as the
enzyme is degrading ACh. The high affinity
AChE blocker physostigmine was used as
reference in our experiments. At 10 mm,
physostigmine induced a constriction of the
preparation even without further EFS (Fig-
ure 4a), which is attributed to a direct
activation of acetylcholine receptors in
addition to AChE inhibition.[20] In contrast,
50 mm AzoTHA in the dark did not evoke
an increase in the basal tone (Figure 4a),
providing a pharmacologically cleaner tool
to study cholinergic systems. When
AzoTHA treated (50 mm) tracheas were
stimulated by EFS (100 Hz, 10 V, 2 ms)
different relaxation kinetics were observed
under irradiation with UV and blue light
(Figure 4b). The observed kinetics (toff) are
in agreement with our previously per-
formed in vitro experiments: In the pres-
ence of cis-AzoTHA (UV irradiation),
AChE is inhibited to a larger extent and
ACh clearance kinetics are significantly
Scheme 1. Design, synthesis, and spectroscopy of AzoTHAs. a) A small compound library of
tacrine-derived photoswitches. b) Three-step synthesis gives the photochromic AChE blocker
AzoTHA, which exhibits reversible cis–trans isomerization using UV light (l=350 nm) and
blue light (l=440 nm). c) UV/Vis spectra of the cis (gray) and trans isomer (blue) of
AzoTHA (inset: expansion of the n–p* band) and THA (black).
AzoTHA-3 showed significantly lower affinities (see the
Supporting Information). As such, trans-AzoTHA is about
ten times less active than THA, but more active than
decamethonium and BisQ.
slower
(toff,cis = (15.25 Æ 1.48) s)
with
Having determined the Ki of AzoTHA, we assessed the
photo-dependent activity of our enzyme inhibitor at this
concentration. In photopharmacology, it is generally advis-
able to employ concentrations close to the Ki (or EC50) of the
dark-adapted state, as the largest dynamic change can be
expected in this region. When we incubated 0.05 UmLÀ1 of
AChE with 1.0 mm of AzoTHA, we were able to induce
photodependent activity (Figure 3a). By contrast, AzoTHA-2
and AzoTHA-3 exhibited no and low photodependent
activity. Our investigations therefore focused on AzoTHA
(Figure 3). Activity was reduced to 17% compared with
unblocked AChE when the sample was irradiated with blue
light (l = 440 nm) and was almost abolished (4% activity)
when the light was switched to UV (l = 350 nm). Thus,
AzoTHA is more active as an inhibitor in its cis state and
becomes less active in the dark-adapted trans state. This effect
could be repeated over many cycles with no apparent loss of
activity (Figure 3b,c). Fitting the off- and on-sets-slopes of
photoswitching exponentially, the ton/off values remain stable
(Figure 3d) over the cycles and their average can be
determined as toff = (4.74 Æ 1.81) s and ton = (4.17 Æ 1.09) s.
To establish that AzoTHA can be used to control
physiological responses with light, we next turned to mouse
trachea. The smooth muscle of this airway constricts in
response to ACh, which can be recorded by force transducers,
an assay known as tracheal tensometry.[18] Electrical field
stimulation (EFS) leads to the release of ACh at the
Figure 3. In vitro photocontrol of AChE with AzoTHA. a) Hydrolysis of
acetylthiocholine in the presence of AChE (0.05 UmLÀ1), AzoTHA (1 mm)
and irradiated with either blue light (l=440 nm, blue bars) or UV light
(l=350 nm, grey bars). b) Reversible photocontrol of AChE (0.05 UmLÀ1
with 1 mm AzoTHA controlled by blue light (l=440 nm, blue bars) and UV
light (l=350 nm, grey bars). Black dots show data points derived from
the initially recorded slope (see a) of catalytic activity, red line shows
binomial fit. c) Average light-dependent activity calculated from b over 5
cycles. d) toff (gray) and ton (blue) values for the repeated deactivation and
activation over several cycles (left side) and their averaged values (right
side). Error bars show standard deviation; N=3 for all experiments.
)
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communication by optical control of an
enzyme that clears a neurotransmitter from
the active zone.
Received: March 24, 2014
Published online: June 4, 2014
Keywords: acetylcholinesterase ·
.
photochromic blockers · photopharmacology ·
tacrine · tracheal tensometry
Figure 4. Ex vivo photocontrol of AChE with AzoTHA. a) Basal tonus is not affected by
AzoTHA (50 mm, black) but with physostigmine (10 mm, red) in mouse trachea
preparations. b) Relaxation kinetics are slower under irradiation with UV light (350 nm)
than with blue light (440 nm) in mouse trachea preparations (50 mm AzoTHA, EFS,
100 Hz, 10 V, 2 ms). c) toff values fitted from (b) with no blocker (black) and with
AzoTHA (50 mm) under blue (l=440 nm, blue) and UV light (l=350 nm, gray); (N=3,
meanÆstandard deviation, t-test: p=0.02).
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blockers. Nevertheless, kinetics under UV and blue light
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most important enzymes involved in synaptic transmission.
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developed a generally applicable method to monitor photo-
chromic AChE inhibitors. Using this in vitro assay, we found
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determined that BisQ, a known photochromic agonists of
acetylcholine receptors, has no photodependent effect on
AChE activity. Further biological evaluation showed that
AzoTHA does have a light-dependent effect on AChE
kinetics in mouse trachea preparations with improved char-
acteristics compared to physostigmine. In accordance with
in vitro data, AChE activity can be reduced to a larger extent
with UV light than with blue light.
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While our paper was in revision, a publication from
Decker and colleagues appeared that demonstrates control of
AChE activity with a dithienylethene photoswitch, allowing
the optical control of b-amyloid aggregation.[21] However,
because of the long irradiation times necessary to achieve
photoswitching and the different biological target, this tool is
not comparable with AzoTHA, which works on a second
timescale and operates on the neuromuscular endplate. Our
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